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. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: Mol Microbiol. 2012 Jul 5;85(3):557–573. doi: 10.1111/j.1365-2958.2012.08127.x

Figure 6. RT-PCR and quantitative real-time PCR analysis of antisense NRG1 production.

Figure 6

A. RNA was isolated from the wild-type (SC5314) and the brg1Δ (ICY326) strains grown under hypha-inducing conditions (YPD+10% FBS, 37°C) for 30 minutes. RNA was treated with DNaseI and used as a template for reverse transcription with either strand-specific primers or oligodT, as indicated above each lane. Different primer combinations (indicated to the right of each panel) were used in PCR reactions with these single stranded cDNA templates. Aliquots of each PCR reaction were electrophoresed on an agarose gel and visualized with ethidium bromide. The housekeeping gene EFB1 serves as a control for the presence of genomic DNA or total cDNA. A no reverse transcriptase sample serves as a control for the RT-PCR. The diagram at the bottom indicates the relative positions of the primers used in this experiment (not to scale). F=NRG1_FOR, S2=NRG1_S2, S=NRG1-S S4=NRG_S4, A=NRG1-A, A2=NRG1_A2, A3=NRG1_A3, A4=NRG1_A4, R=NRG1_REV, dT=oligodT, G=gDNA, N=no reverse transcriptase control.

B. The wild-type (dark bars) and the brg1Δ (light bars) strains were grown under hypha-inducing conditions (YPD+10% FBS, 37°C). Samples were removed prior to, as well as 20 and 30 minutes after induction and cDNA synthesized with oligodT for ACT1 and BRG1 real-time PCR reactions, and NRG1_S2 and NRG1_REV for the antisense and sense reactions, respectively. Levels were normalized by ACT1 and expressed relative to the yeast control culture (wild-type time 0 sample, therefore its results are 1.0). Error bars represent the standard error of the mean. BRG1 expression is rapidly upregulated in the early stages of hyphal induction (upper panel). The NRG1 sense transcript falls in the wild-type, but remains constant in the brg1Δ strain. Conversely, the antisense NRG1 transcript rises concurrently with BRG1 in the wild-type (SC5314) but remains similar to time 0 levels in the brg1Δ strain (ICY326).

C. BRG1 expression does more than stimulate antisense NRG1 production. In yeast conditions, the nrg1Δ strain (BCa23-3) is constitutively pseudohyphal. Strains over-expressing BRG1 (ICY171 and ICY175), however, form hyphae under the same conditions. Cell walls were visualized with calcofluor white and nuclei with DAPI.